Ribonucleases such as ribonuclease A (“RNase A”) and their cytotoxicity towards tumor cells were discovered in the 1960s (reviewed in Roth, J., Cancer Res. 23:657–666 (1963)). In the 1970s, human serum was also discovered to contain several RNAses that are expressed in a tissue specific manner (Reddi, E., Biochem. Biophys. Res. Commun. 67:110–118 (1975); and Blank, et al., HUMAN BODY FLUID RIBONUCLEASES: DETECTION, INTERRELATIONSHIPS AND SIGNIFICANCE, pp 203–209 (IRL Press, London, 1981)).
Further to these early studies was the discovery that an anti-tumor protein from oocytes of Rana pipiens had homology to RNAse A (Ardelt, et al., J. Biol. Chem. 256:245–251(1991)). This protein was termed ONCONASE®, Alfacell Corporation, N.J. See also e.g., Darzynkiewicz, et al., Cell Tissue Kinet. 21:169–182 (1988); Mikulski, et al., Cell Tissue Kinet. 23:237–246 (1990); and U.S. Pat. No. 4,888,172).
Phase I and Phase I/II clinical trials of ONCONASE® as a single therapeutic agent in patients with a variety of solid tumors (Mikulski, et al., Int. J. of Oncology 3: 57–64 (1993)) or combined with tamoxifen in patients with advanced pancreatic carcinoma have been completed (Chun, et al., Proc. Amer. Soc. Clin. Oncol. 14:210 (1995)) and the protein has been found to be efficacious in pancreatic, renal cell, and prostate cancers as well as mesothelioma.
Conjugation of ONCONASE® to cell-type-specific ligands was found to increase its potency towards tumor cells (Rybak, et al., Drug Delivery 1:3–10 (1993)). Taken together, these results indicated that ONCONASE® has properties advantageous to the generation of a potent selective cell killing agent.
Development of ONCONASE® conjugates for human therapeutics has been slow. ONCONASE® is derived from amphibian tissue and trace contaminants present in the purified preparation stimulate undesirable immune responses in humans. This side-effect has led to production of a recombinant form of the protein (Newton, et al., Protein Engineering 10:463–470 (1997) and PCT published application WO 97/38112).
However, expression of active recombinant ONCONASE® has been problematic. ONCONASE® requires a pyroglutamic acid at the N-terminus for activity. Unfortunately, ONCONASE® with a N-terminal glutamine is not expressed by bacteria but accumulates in insoluble inclusion bodies. To increase bacterial expression of soluble ONCONASE®, methionine has been appended to the N-terminus. However, this modification of the protein prevents the formation of the pyroglutamic acid necessary for activity. Therefore, it has been necessary to engineer ONCONASE® with an N-terminal methionine only to remove it for activity. The cleaved and the uncleaved proteins must then be separated to obtain a pure composition of high purity and activity.
Other problems have arisen in the manufacture of ONCONASE®-based fusion proteins. It has been difficult to fuse recombinant ONCONASE® in frame to ligand binding moieties and retain proper folding of both the ONCONASE® and the ligand binding moiety. This has limited the use of ONCONASE® in targeted cell killing to only those compounds that can be chemically conjugated.
Thus, there exists in the art a need for recombinant ribonucleases that can be expressed in bacteria and retain activity. Furthermore, there exists a need for a ribonuclease with anti-tumor activity that retains its activity when produced as a single chain fusion protein. This invention fulfills these and other needs.